The present invention relates to a process for the preparation of a vaccine against tuberculosis and other intracellular pathogens. This vaccine is targeted against intracellular pathogens, more particularly the pathogen Mycobacterium tuberculosis and Salmonella in this case.
The utility of the present invention is to develop a vaccine against the intracellular pathogens, which are causative agents of tuberculosis, brucellosis, leishmaniasis, listeriosis, leprosy, malaria, typhoid, trypanosomiasis and streptococcus and HIV-infection. The pathogen Mycobacterium tuberculosis (M. tuberculosis) the subject matter of this invention is a causative agent of tuberculosis. In this invention M. tuberculosis was allowed to grow in the allogeneic and syngeneic macrophages and macrophage cell lines. The macrophagesxe2x80x94M. tuberculosis complex was then irradiated to kill the macrophages as well as the mycobacterium.
Tuberculosis is a chronic infectious disease that continues to kill some 3 million people a year. About 8 million new cases arise every year and the number continues to increase. About one-third of the world population is infected with M. tuberculosis. The emergence of AIDS has reactivated tuberculosis in millions of dormant individuals, causing a sharp rise in the number of cases and deaths. M. tuberculosis is therefore responsible for the highest morbidity rate among all infectious agents. The only available vaccine BCG is both unpredictable and highly variable. Doubtful efficacy of BCG vaccination has put the scientific community to urgently develop effective means of vaccination against the M. tuberculosis (Bloom, B. R. et. al., Annu. Rev. Immunol. 10:1992:453).
During the past many decades BCG has been extensively used as a vaccine world over. Several hundred million children and new born have been the recipient of BCG vaccine. However, in spite of wide usage of BCG vaccine, tuberculosis has still become the fastest spreading disease not only in developing countries but also in the industrialized world. Further, the protective efficacy of the current BCG vaccine is both unpredictable and highly variable and it remains the most controversial of all currently used vaccines. Its doubtful efficacy in controlled trials have increased the concern about its use as a vaccine (Bloom and Fine, Tuberculosis In B. Bloom (ed.), 1994:531, Bloom, B. R. et. al., Annu. Rev. Immunol. 10:1992:453). Furthermore, the extensive clinical trials done in Madras showed similar extent of protection in BCG-vaccinated and unvaccinated individuals, indicating that BCG induced zero protection (Ind. J. Med. Res. 1980:72(Suppl.):1-74). Thus it is obvious that BCG vaccination does not prevent transmission.
In past also, many questions always arose pertaining to the safe use of BCG vaccine.
A major catastrophe that cast a cloud over the reputation of BCG vaccination occurred in 1929. In Lubeck, Germany, 251 children received a BCG vaccine prepared at a local institute, and 72 of these children died. Subsequent investigation revealed that the institute also maintained cultures of virulent tubercle bacilli and that the batch of BCG vaccine given to the children had accidentally been contaminated with one of these strains of Mycobacterium tuberculosis (Lubeck. 1935. Die Sauglingstuberkulose in Lubeck. Springer, Berlin).
A new question has arisen regarding the safety of BCG in HIV-infected individuals. A small number of cases of disseminated BCG-osis have been reported among children who received BCG vaccine and were subsequently found to be HIV seropositive (Von Reyn, et. al. Lancet 1987: ii:669-672; Braun, et. al., Pedietr. Infect. Dis. J. 1992:11:220-227; Weltman, et. al., AIDS 7:1993:149). WHO currently recommends discontinuing the use of BCG vaccine in children showing overt signs of immunodeficiency (World Health Organization, 1992, Expanded Program for Immunization, Program Report,World Health Organization, Geneva; Weekly Epidemiol. Rec. 62:1987:53).
Large volunteer studies by Dahlstrom and Difts (Scand J Respir Dis Suppl. 65:1968:35) and a meta-analysis of BCG in the prevention of tuberculosis based on 13 prospective studies and 10 case control studies has recently been completed (Colditz et. al., J. Amer. Med. Assoc. 271:1994:698-702). While it concluded that on average BCG was about 50% protective in preventing tuberculosis, the biological and operational significance of averaging, in essence, such widely divergent results are itself arguable.
Before the advent of AIDS, in most wealthy countries, the incidence of tuberculosis was declining for at least a century. This is illustrated in comparisons between The Netherlands (which never employed BCG vaccination) and the United Kingdom and Scandinavia (which instituted national BCG vaccination in the 1950). The declines in tuberculosis cases reported in these countries were similar (Styblo, K., Selected Papers R. Netherland Tuberc. Assoc. 24:1991:136; Sutherland, Bull. Int. Union. 57:1981:17). Thus it is unreasonable to attribute that the decline was due to BCG vaccination alone.
BCG""s performance is based on a hypothesis that BCG is effective against primary infection in children and endogenous reactivation of long-standing infections but not against exogenous infection (ten Dam, H. G. Adv. Tuberc. Res. 21:1984:79; ten Dam, H. G. and A. Pio. Tubercle 63:1988:226). Epidemiological data suggest that BCG vaccination imparts greater or more consistent protection against systemic disease, in particular miliary tuberculosis and tuberculosis meningitis in children, than against pulmonary disease (Rodrigues, et.al., Int J epidemiol. 22:1993:1154). Lurie""s studies indicated that the number of CFU of M. tuberculosis isolated from lungs of BCG-immunized versus unimmunized rabbits showed no difference in the number of organisms reaching and capable of being cultured from lung and other tissues.
Another insight is provided by the intracellular location of the mycobacterium. Electron microscopic findings indicate that BCG remains essentially entirely within the phagolysosomes after in vitro infection of macrophages, whereas virulent M. tuberculosis (strain H37Rv) can escape from the phagolysosome and enter the cytoplasm (McDonough, et.al., Infect. Immun. 61:1993:2763). This may be relevant insofar as it is the antigens in the endosomal compartment of antigen-presenting cells that are presented in conjunction with MHC class II determinants to CD4+ T helper cells, whereas cytoplasmic antigens are presented in association with the Major Histocompatibility Complex (MHC) class I determinants to CD8+ Cytotoxic T cells (CTL). If these findings in vitro are general, they will explain why M. tuberculosis is more dependent for its elimination on MHC class I-restricted CTL than BCG and suggests that BCG may not be very effective in eliciting MHC class I-restricted CTL (Stover, et.al., Nature 351:1991:456). In this context, Rich, 1951 (The Pathogenesis of Tuberculosis, 2nd, p. 1028; Charles C Thomas, Publisher, Springfield, Ill.), Canetti, 1955 (The Tubercule Bacilli in the Pulmonary Lesion of Man, p. 226; Springer, N.Y.) and Lurie, 1964 (Resistance to Tuberculosis. Experimental Studies in Native and Acquired Defense, p. 391; Harvard University Press, Cambridge Press, Cambridge, Mass.), commented that recovery from infection with M. tuberculosis provided stronger protection against future tuberculosis than could BCG.
The effective resistance to M. tuberculosis infection will require participation both of specific CD8+ CTL to lyse macrophages or parenchymal cells unable to restrict their infection and of specific CD4+ T cells able to produce IL-2, IFN-xcex3, TNF-xcex1, and other lymphokines involved in macrophage activation.
Considering these drawbacks of the BCG-vaccine, the applicants have taken advantage of the fact that the vaccine will be used as an irradiated preparation and has no fear of inoculating in AIDS patients and immunocompromised children. BCG is given as an attenuated preparation and is not recommended in these subjects because it causes disseminated BCG-osis, WHO currently recommends discontinuing the use of BCG vaccine in children showing overt signs of immunodeficiency (World Health Organization. 1992. Expanded Program for Immunization. Program Report. World Health Organization, Geneva. World Health Organization. Weekly Epidemiol. Rec. 1987:62:53-54).
Another insight is provided by the intracellular location of the mycobacterium. BCG remains essentially entirely within the phagolysosome of macrophages, whereas virulent M. tuberculosis can escape from the phagolysosome and enter the cytoplasm (McDonough, K. A., Y. Kress, and B. R. Bloom. 1993. Infect Immun. 61:2763-2773). The antigens in the endosomal compartment of antigen-presenting cells are presented in conjunction with MHC class II determinants to CD4+ T helper cells, whereas cytoplasmic antigens are presented in association with the Major Histocompatibility Complex (MHC) class 1 determinants to CD8+ Cytotoxic T cells. M. tuberculosis is more dependent for its elimination on MHC class I-restricted CTL. BCG is not effective in eliciting MHC class I-restricted CTL (Stover, et.al., Nature 351:1991:456). The present vaccine contains the irradiated preparation of M. tuberculosis grown in macrophages. M. tuberculosis infected macrophages are reported to effectively generate CTL (Stover, et.al., Nature 351:1991:456). Further, it has also been reported that irradiated cells undergo apoptosis and can be phagocytosed by the dendritic cells (Albert, M. L., et.al., Nature 392:1998:86) and it leads to the generation of antigen specific CD4+ and CD8+ T cell response. This apoptosis-dependent pathway may not only have potential in vaccination studies but also for therapeutically manipulating immune system to induce T-helper and CTL response in vivo to a variety of antigens including tumor, and possibly to modulate favourable immune response.
Rich (The pathogenesis of Tuberculosis. 2nd ed, p. 1028, 1951. Charles C Thomas, Publisher, Springfield, Ill.), Canetti (The tubercle Bacillus in the pulmonary Lesion of Man, p. 226, 1955. Springer, N.Y.), and Lurie (Resistance to Tuberculosis. Experimental Studies in Native and Acquired Defense, 391, 1964. Harvard University Press, Cambridge, Mass.) have commented that recovery from infection with M. tuberculosis provided stronger protection against future tuberculosis than could BCG. In context with the above statements, the candidate vaccine has advantage over existing BCG vaccine because it contains the M. tuberculosis grown in the natural environment of the macrophages that secrete the unique antigens responsible for the induction of protective immune response and can generate CD4+ T-helper cells and CD8+ CTL. The effective resistance to M. tuberculosis infection will require participation of both specific CD8+ CTL to lyse macrophages or parenchymal cells unable to restrict their infection and of specific CD4+ T cells able to produce IL-2, IFN-xcex3, TNF-xcex1, and other lymphokines involved in macrophage activation.
The main rationale behind this process was to develop a vaccine against tuberculosis and other intracellular diseases, MHC-matched (syngeneic) and mismatched (allogeneic) macrophages harboring M. tuberculosis on irradiation undergo apoptosis; dendritic cells engulf these macrophages and present the antigen (Mycobacterium-proteins and allo-macrophage peptides) on their surface and induce naxc3xafve T-cells to differentiate into effecter CD4* Th1 cells. These dendritic cells also activate CD8* T cells for cell-mediated immunity. Allo-macrophages in the system generate an allo-reaction and as a result a large amount of cytokines like IL-2, IL-12, IFN-xcex3, etc., are produced which promote the Th1 response and cell mediated immune response. It is known that Th1-type of response provides protection against tuberculosis. Hence the main utility of the process was to produce a potent and specific vaccine against M. tuberculosis. 
The main object of the present invention thus is to develop a vaccine against tuberculosis and other intracellular diseases like leprosy, leishmaniasis, typhoid, trypanosomiasis malaria, brucellosis, listeriosis, AIDS, streptococcal infection and cancer.
Another object of the present invention is to culture the pathogen inside the syngeneic and allogeneic macrophages and allow them to secrete antigens within the cells.
Yet another object is to develop a method whereby the pathogen are killed by the already known drugs and further will be gamma irradiated before use; the gamma irradiated cells are known to undergo apoptosis and are engulfed by the dendritic cells. Dendritic cells are potent activator of Th1 cells and CD8+ cytotoxic cells.
Another object is to develop a vaccine that acts against both syngeneic macrophages entrapped pathogens (viz. M. tuberculosis, M. leprae, leishmania, salmonella, trypanosoma, malaria, brucella, listeria, HIV, streptococcus) (e.g. SMTV, S=syngeneic, M=macrophage, T=tuberculosis, V=vaccine) and allogeneic-macrophages entrapped pathogen vaccine (e.g., AMTV, A=allo, M=macrophage, T=tuberculosis, V=vaccine), to generate protective immune response.
Still another objective of the present invention is to develop a vaccine based on entrapment of pathogen in the allogeneic cells that would elicit immune response irrespective of the genetic background i.e. it will work as a promiscuous vaccine, and hence it will work irrespective of the genetic diversity in the human subjects.
The present invention relates to a process for the preparation of a vaccine against tuberculosis and other intracellular pathogens. This vaccine is targeted against intracellular pathogens, more particularly the pathogen Mycobacterium tuberculosis and Salmonella in this case.